Dynamic phase-modulation, fluorescence-based sensors are generally known. Instruments of this type are, for example, being developed or proposed for use in hospitals to monitor the concentration of gases such as oxygen, ionized hydrogen and carbon dioxide within the blood of patients. The substance of interest (e.g., oxygen) is known as the analyte.
A known property of fluorescent and photoluminescent substances referred to as fluorophores is that they absorb energy and are driven from their ground state energy level to an excited state energy level in response to the application of energy from a light source. The fluorophores are unstable in their excited states, and fluoresce (radiative decay) or give off excess thermal energy (non-radiative decay) as they return to their ground state. The fluorescence lifetime, .tau., represents the average amount of time the fluorophore remains in its excited state prior to returning to the ground state. The fluorescence intensity, I, represents the intensity of the emission given off by the fluorophore as it returns to the ground state.
When a fluorophore in a sensing element is photoexcited in the presence of another diffusing substance known as a quencher, Q, collisional interactions between the excited state and the quencher introduce a new mechanism for non-radiative decay, resulting in a decrease in both the fluorescence intensity and excited state lifetime. This process is known as dynamic fluorescence quenching. Furthermore, the amount by which the intensity and lifetime decrease from the respective intensity, I.sub.o, and lifetime, .tau..sub.o, in the absence of quencher is directly related to the quantity of the quencher, [Q], present in the sensing element to which the fluorophore is exposed while fluorescing. The relationship between the fluorescence intensities and lifetimes in the absence and presence of quencher is described by the Stern-Volmer equation: EQU I.sub.o /I=.tau..sub.o /.tau.=1+k.sub.q .tau..sub.o [Q]
where:
I.sub.o is the fluorescence intensity in the absence of quencher; PA1 I is the fluorescence intensity in the presence of quencher; PA1 .tau..sub.o is the excited state lifetime in the absence of quencher; PA1 .tau. is the lifetime of the fluorophore in the presence of quencher; PA1 k.sub.q is the bimolecular quenching rate constant in the sensing element; and PA1 [Q] is the concentration of the quencher in the sensing element. PA1 K.sub.SV =ak.sub.q .tau..sub.o ; PA1 a=[O.sub.2 ]/P.sub.O2 PA1 [O.sub.2 ] is the concentration of oxygen in the sensing element and; PA1 P.sub.O2 is the partial pressure of oxygen in the medium being sensed.
Measurements of intensity or lifetime can be used to determine the concentration of a quencher in a sensing element, for example oxygen in a sensor for blood oxygen determination. The oxygen concentration in the sensing element, [O.sub.2 ], can be related to the oxygen partial pressure in the blood stream, P.sub.O2, by a solubility constant, a. The general form of the Stern-Volmer equation therefore becomes: EQU I.sub.o /I=.tau..sub.o /.tau.=1+ak.sub.q .tau..sub.o P.sub.O2 =1+K.sub.SV P.sub.O2
where:
Oxygen is often both the analyte and fluorescence quencher for the sensing element, although this is not and need not always be the case. The fluorescence quencher and analyte of interest may be different substances, but the concentration of the analyte of interest is related to the quencher concentration by a known relationship. For example, the Moreno-Bondi et al. article Oxygen Optrode for Use in a Fiber-Optic Glucose Biosensor, Analytical Chemistry, Vol. 62, No. 21, (Nov. 1, 1990) describes a glucose sensor based on glucose dependent consumption of oxygen catalyzed by the enzyme glucose oxidase. The glucose oxidase is immobilized onto the surface of an oxygen sensing element. As the concentration of glucose in the external medium increases, more oxygen is consumed within the sensing element, resulting in a change in the dynamic quenching of the fluorescence by oxygen. The analyte concentration is therefore computed as a function of the quencher concentration actually measured by the instrument.
For purposes of this patent document, the term "quencher" is used to refer to the actual fluorescence quenching substance, whether this quencher is the analyte of interest or another substance related to the analyte of interest by a known relationship. It is to be understood that the concentration of the analyte of interest can be determined on the basis of the measured concentration of the quencher and the known relationship between the analyte and quencher.
A well recognized advantage of determining the fluorescence lifetime in optical sensing applications is that it is insensitive to dye concentration, optical coupling efficiencies and lamp variations.
Phase-modulation fluorescence spectroscopy is a known method for determining the lifetime, .tau., of a fluorophore. During the phase-modulation method the medium containing the analyte is excited by a light beam or other excitation signal that is preferably sinusoidally amplitude modulated at a radial frequency .omega.=2.pi.f, where f is the frequency in cycles per second. The fluorescence emission from the fluorophore is a forced response to this excitation signal, and is therefore amplitude modulated at the same radial frequency .omega. as the excitation signal. However, because of the finite lifetime of the fluorophore in the excited state, the emission is phase shifted by an angle .THETA. with respect to the excitation signal. Furthermore, the amplitude or intensity of the emission is less modulated (demodulated) by an amount m with respect to the excitation signal. The lifetime of the fluorophore can be calculated in a known manner from measurements of the phase shift (tan .THETA.=.omega..tau.) and demodulation factor (m=(1+.omega..sup.2 .tau..sup.2).sup.-1/2).
Known phase-modulation, fluorescence-based sensing instruments such as those described in the Dukes et al. U.S. Pat. No. 4,716,363 and the Barnes et al. Canadian Patent Application 2,000,303 make use of the phase-modulation technique described above (and are operated "univariantly" as herein described) to measure the concentrations of analyte such as the partial pressure of oxygen, in a medium such as blood. These instruments include an optical fiber sensing element which is connected at the proximal end to both an optical excitation system and an optical detector. The distal end or tip of the optical fiber includes a polymer matrix which is permeable to the quencher and includes one or more fluorescable indicator components in the form of dyes. For in-vivo sensors, the sensing element is configured in the form of a probe or catheter insertable into a blood vessel of a patient to provide on-line monitoring of the oxygen concentration.
In phase modulation fluorescence spectroscopy the modulation frequency is typically chosen such that .omega..tau..apprxeq.1 within the range of analyte concentration of interest. This corresponds to a phase angle near 45 degrees, where a calculation of the lifetime is least sensitive to small errors in the measured phase shift and measured demodulation ratio. The Holst et al. article Oxygen-Flux-Optode for Medical Application, Proc. SPIE-Int. Soc. Opt. Eng., V.1885 (Proceedings of Advances in Fluorescence Sensing Technology, 1993) pages 216-227, and Bacon et al. U.S. Pat. No. 5,030,420 describe oxygen sensors in which the phase shift is measured as a function of oxygen concentration at a fixed modulation frequency. The phase information is used to determine the fluorescence lifetime as a function of oxygen concentration. The Holst et al. article describes a sensor for which the excited state lifetime of the fluorophore in the absence of quencher is 205 nsec, from which they determine an optimum modulation frequency f.sub.opt =777 kHz (.omega..tau..apprxeq.1). For similar reasons, "univariant" sensing instruments are typically configured in such a manner that .omega..tau..apprxeq.1 or .theta..apprxeq.45.degree. over the concentration range of interest. For example, the Dukes et al. U.S. Pat. No. 4,716,363 describes a feedback system which provides the modulation frequency required to give a constant phase shift of about 45 degrees. The resulting frequency is used to determine the excited state lifetime as a function of analyte concentration. Dukes et al. suggests that the constant phase angle approach offers an advantage in that .omega..tau.=1 for all analyte concentrations of interest.
As discussed by Wolfbeis in the book Fiber Optic Chemical Sensors and Biosensors, Vol. II, CRC Press 1991 and taught by Mauze et al. in U.S. Pat. No. 5,057,277, when using intensity or lifetime measurements to determine analyte concentration, too large a Stern-Volmer quenching constant K=ak.sub.q .tau..sub.o can be undesirable. In particular, when the quenching constant is too large, relatively large changes in lifetime and intensity occur over a narrow range of analyte concentrations. At larger analyte concentrations of interest, analyte dependent changes in the fluorescence intensity and lifetime become undesirably small. These considerations are relevant, for example, in the proper design of a sensor for monitoring oxygen partial pressure in blood, where accuracy is desired over the range of P.sub.O2 =40-120 mm mercury, more preferably over the range of P.sub.O2 =40-200 mm mercury. A minimum accuracy of about 3 mm (about 2%) is typically required over this range.
When configured for insertion into the body of a patient, the sensing element can be required to be as small as 125 .mu.m in diameter. The amount of luminescent dye that can be accommodated on the tip of the sensing element is therefore limited, resulting in a relatively low fluorescent signal. This problem can be partially countered by increasing the dye loading and/or photon flux. Unfortunately, dyes suitable for fluorescence quenching sensors are often subject to dye aggregation and photobleaching. Aggregation and/or photobleaching often cause the indicator components in the dye to exhibit a number of different lifetimes .tau..sub.o, i.e., heterogeneous lifetimes. For any given sensor, the heterogeneous lifetimes often vary with aging and/or photodegradation. Manufacturing process variables can also result in variable lifetimes from sensor to sensor.
Calibration plots based on a measurement of the excited state lifetime will necessarily vary with changes in .tau..sub.o, regardless of the method used to determine the lifetimes. Typically, two point sensor calibration and recalibration procedures are required. The initial calibration is typically performed following the assembly of the sensor or immediately prior to use. Recalibration is often required while the sensor is in use to maintain measurement accuracy. Two point, calibration procedures involve the use of two calibrants, each having known analyte concentrations. The calibrants will typically have known analyte concentrations close to the maximum and minimum concentrations of the range over which measurements are taken. During two point calibration procedures, the sensing element is alternately exposed to the two calibrants, and the slope and intercept of a calibration plot is determined so that the sensor can provide accurate analyte concentration readings. In effect, two point calibration involves adjusting the slope and intercept of the calibration data, as represented by the lookup table data or mathematical equation stored in memory of the sensor processor, until the relationship characterized by the data extends through the points corresponding to those of the known calibrants.
Since two point calibration procedures require the sensing element to be exposed to two calibrants, both in-vivo and ex-vivo sensors must be removed from the a-line circuit of arterially catheterized patients for recalibration. However, this is not acceptable procedure in most clinical situations since it can compromise the patient by, for example, increasing the risk of infection. Two point calibration procedures are also relatively time consuming.
It is evident that there is a continuing need for improved phase-modulation, fluorescence-based sensing instruments. In particular, there is a need for sensors of this type having calibration plots with slopes or slopes and intercepts that are insensitive to drift and instability caused by heterogeneous lifetimes and other variations in lifetime. A sensor capable of being characterized by a calibration plot having slopes which are insensitive to .tau..sub.o variability would have the capability of being calibrated with the use of only one calibrant having a known analyte concentration. Slopes would not depend on .tau..sub.o variability associated with unwanted dye aggregation during sensor manufacture. Furthermore, any differential photodegradation of the larger .tau..sub.o species in a sensor would give rise to a change in the intercept, but not the slope of the calibration plot. Such a sensor capable of single point calibration would increase the commercial acceptance of in-vivo blood gas monitoring since it would enable in-vivo recalibration.